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Abstract

Protease-activated receptors (PAR1-4) are activated by proteases released by cell
damage or blood clotting, and are known to be involved in promoting pain and hyperalgesia.
Previous studies have shown that PAR2 receptors enhance activation of TRPV1 but the
role of other PARs is less clear. In this paper we investigate the expression and
function of the PAR1, 3 and 4 thrombin-activated receptors in sensory neurones. Immunocytochemistry
and in situ hybridization show that PAR1 and PAR4 are expressed in 10 - 15% of neurons,
distributed across all size classes. Thrombin or a specific PAR1 or PAR4 activating
peptide (PAR1/4-AP) caused functional effects characteristic of activation of the
PLCβ/PKC pathway: intracellular calcium release, sensitisation of TRPV1, and translocation
of the epsilon isoform of PKC (PKCε) to the neuronal cell membrane. Sensitisation
of TRPV1 was significantly reduced by PKC inhibitors. Neurons responding to thrombin
or PAR1-AP were either small nociceptive neurones of the peptidergic subclass, or
larger neurones which expressed markers for myelinated fibres. Sequential application
of PAR1-AP and PAR4-AP showed that PAR4 is expressed in a subset of the PAR1-expressing
neurons. Calcium responses to PAR2-AP were by contrast seen in a distinct population
of small IB4+ nociceptive neurones. PAR3 appears to be non-functional in sensory neurones. In a
skin-nerve preparation the release of the neuropeptide CGRP by heat was potentiated
by PAR1-AP. Culture with nerve growth factor (NGF) increased the proportion of thrombin-responsive
neurons in the IB4- population, while glial-derived neurotropic factor (GDNF) and neurturin upregulated
the proportion of thrombin-responsive neurons in the IB4+ population. We conclude that PAR1 and PAR4 are functionally expressed in large myelinated
fibre neurons, and are also expressed in small nociceptors of the peptidergic subclass,
where they are able to potentiate TRPV1 activity.

Introduction

Proteases released during injury activate protease-activated receptors (PARs), a family
of four G protein-coupled receptors, by cleaving the extracellular N-terminal domain
to expose a tethered peptide ligand [1-5]. PAR1, PAR3, and PAR4 are activated by thrombin, reviewed in [5,6], while PAR2 is not activated by thrombin but is activated by trypsin and mast cell
tryptase [7-9]. PAR4 is specifically activated by cathepsin G [10].

In sensory neurons of the dorsal root ganglia (DRG) a functional response to thrombin
was initially reported by Gill et al [11]. The mRNA of all four PARs is expressed in sensory neurons [12]. There is clear evidence for the functional involvement of PAR2 receptors in peripheral
mechanisms of inflammation and pain [13-15], partly via sensitisation of the transient receptor potential vanilloid subfamily
1 (TRPV1) receptor [15-18] and partly by stimulating the release of substance P and CGRP from the terminals
of afferent neurons [13,19,20]. Sensitization of TRPV1 depends on activation of the epsilon isoform of PKC (PKCε),
which can be observed as a translocation of PKCε from the cytoplasm to the surface
membrane [21], and a similar translocation has been reported in response to activation of PAR2
[22].

Thrombin is released by blood clotting following blood vessel damage or tissue injury,
and can act on PAR1, 3 and 4 expressed in primary sensory nerve terminals present
in the vicinity. Thrombin injected into peripheral tissues induces proinflammatory
effects, such as protein extravasation and vasodilation, which are mediated at least
in part by a neurogenic mechanism [9,14,23]. Activation of PAR1 may be involved in peripheral nerve damage [24,25]. Some reports, however, describe antinociceptive effects of activation of peripheral
PAR1 activation with subinflammatory protease concentrations [26,27]. PAR4 activation has also been shown to be analgesic [28-30], but other evidence shows that the administration of a PAR4 activator peptide (PAR4-AP)
causes the formation of edema and leukocyte recruitment in a rat paw model of inflammation
[31].

To the best of our knowledge no studies have investigated the localization of functional
PAR1, 3 and 4 receptors in sensory neurons, nor the role of receptors activated by
thrombin in TRPV1 sensitisation or in activation of PKCε in nociceptors. These questions
are addressed in the present study. We initially compared the effects of thrombin
in adult and neonatal rats and mice in order to compare PAR functional expression
in different species and ages. In fact, though, we saw few qualitative or quantitative
differences between these four groups of animals in responses to PAR activation. Most
experiments were therefore continued in neurons from adult mice only, which also gave
us the opportunity to compare the results in wild-type and transgenic animals in which
the roles of specific PAR receptors were explored by deletion of PAR1 or PAR2.

Electrophysiology

Methods used were as described before [32-34]. In brief, all recordings were made from the somata of DRG neurons with the whole
cell patch-clamp method, at a holding potential of -70 mV, using an Axopatch 200B
amplifier and pClamp software (Molecular Devices, Palo Alto, CA). Test solutions were
applied using a multibarrel automated rapid solution changer (CVscientific, University
of Modena, Modena, Italy). Only one recording was performed on each culture dish to
ensure that data were not obtained from cells that had been inadvertently exposed
to other test treatments. All experiments were performed at room temperature (20 -22°C).

Immunohistochemistry: DRG sections

DRGs from adult male TO mice (25-30 g, Tucks, UK) were rapidly removed and post-fixed
in 10% formalin solution for 72 h, embedded in paraffin, sectioned at 4 μm on a sledge
microtome (Leitz, Nussloch, Germany) and mounted on Fisher Superfrost/Plus slides
(BDH, UK). Sections were dewaxed in xylene, incubated in 0.3% hydrogen peroxide in
methanol to quench endogenous peroxidase activity and hydrated through an ethanol
series. Sections were then blocked in 5% normal goat serum in 0.01 M PBS containing
0.03% Triton X-100, prior to overnight incubation at +4°C with the respective antibodies.
Affinity-purified goat polyclononal IgGs (concentration 200 μg/ml) were obtained from
Santa Cruz Biotechnology Inc (California, USA) and had been previously characterised
by Western blot and immunohistochemistry. Anti-PAR1 (sc-8204; 1/100 dilution) was
raised against a peptide mapping at the N-terminal of mouse PAR1, and reacts with
PAR1 of mouse and rat origin. Anti-PAR3 (sc-8209; 1/800) was raised against a peptide
mapping to the C-terminus of mouse PAR3, and reacts with PAR3 of mouse and rat origin.
Anti-PAR4 (sc-8462; 1/150) was raised against a peptide mapping at the C-terminal
of PAR4 of mouse origin, and reacts with PAR4 of mouse and rat origin. Immunoreactivity
was detected using biotinylated donkey anti-goat secondary antibodies raised against
the primary antibody host (7.5 μg/ml, Vector Laboratories, Peterborough, UK), followed
by avidin-biotin complex (ABC) (Vector Laboratories, UK) and subsequently visualised
using diaminobenzidine (DAB)/hydrogen peroxide (Biogenex, Finchampstead, UK). All
immunohistochemical detection steps (from secondary antibody stage onwards) were performed
on an Optimax (Biogenex, UK) robotic immunostainer to increase intersection staining
consistency, thereby increasing the accuracy and reliability of semiquantitative analysis.
Sections were counterstained with Gill's haematoxylin followed by acid-alcohol (0.5%
concentrated hydrochloric acid in 70% ethanol). Control experiments for immunohistochemistry
were performed by incubation with normal goat serum in place of primary antibodies
and resulted in a complete absence of staining (not shown). Specific labelling was
tested by incubation of sections with affinity-purified antisera and a 20-fold excess
of peptide obtained from Santa Cruz Biotechnology Inc (California, USA) and corresponding
to the antigenic sequence to which the antisera were raised. Antibody blocking in
this way resulted in a complete absence of specific staining (not shown), though background
levels were similar to those shown in Fig. 1.

In situ hybridisation (ISH)

The DRGs of 25-30 g adult male TO mice were rapidly removed after cervical dislocation,
frozen in isopentane, chilled to -40°C and sectioned at 10 μm using a Brights cryostat
(model OTF). Sections were post-fixed with 4% paraformaldehyde in PBS, pH 7.2, dehydrated
through an ethanol series and stored in 95% ethanol at 4°C until use. Oligonucleotide
probes specific to mouse PARs were designed (see Table 1) and custom-synthesised by Sigma Genosys (Cambridge, UK). Purification was by 8M
urea/8M polyacrylamide preparative sequencing gel electrophoresis. Specificity was
thoroughly checked using BLAST. For PAR1, the probe was synthesised complementary
to bases 969-1008 (according to GenBank Acc. No L03529); for PAR2, complementary to bases 930-969, according to [35]; for PAR3, complementary to bases 351-390, according to [36]; and for PAR4, complementary to bases 1059-1098, according to [37,38].

The cross sectional areas of neuronal profiles with a visible nucleus were measured
using the Scion Image Analysis system. Silver grains overlying each identified neuronal
profile were counted for PAR1, PAR2 and PAR4. For PAR3 the high levels of expression
of this receptor's mRNA made grain counting impossible, and cells were given a score
(-, negative/below detectable levels; +, weakly labelled; ++ moderately labelled;
+++, intensely labelled). Signal intensity for PAR1, PAR2 and PAR4 was determined
by dividing grain counts by the area of the neuronal profile. To reduce the risk of
biased sampling of the data owing to varying emulsion thickness and background density
of silver grains for each section, a signal/noise (S/N) ratio was used, as described
previously [40]. The signal intensity of each neuronal profile was expressed as a S/N ratio of the
mean background level as described [41,42]. Neuronal signal intensities greater or equal to three times the background level
(S/N≥3) were considered positively labelled.

Immunocytochemistry: isolated DRG neurons and glia

For PKCε visualization, rat DRG neurons cultured for 1-3 d in vitro were treated with
a PAR agonist (for times see Table 2) and then rapidly fixed for 10 min at room temperature with paraformaldehyde/PBS
(4% formaldehyde and 4% sucrose mixed 50:50 with PBS). Fixed cells were washed three
times in PBS (with 0.1% fish skin gelatin to block nonspecific binding), permeabilized
for 30 min at room temperature with Triton X-100 (0.2% in PBS), and incubated overnight
at 4°C with rabbit polyclonal anti-PKCε antibody [33] diluted 1:1000 in PBS-T/gelatin (PBS with 0.05% Triton X-100). Coverslips were then
incubated for 1 h at room temperature with donkey anti-rabbit IgG conjugated to the
fluorophore Alexa Fluor 488 (1:200; Invitrogen), washed three times in PBS/gelatin,
and visualized with a confocal microscope (Leica SP2).

To characterize subpopulations of protease-responsive neurons, double immunostaining
on DRG cultures was performed. Coverslips processed for PKCε immunoreactivity as above
were incubated overnight at 4°C with the following polyclonal antibodies: anti-substance
P (SP), anti-calcitonin gene-related peptide (CGRP), anti-N52 (1:100; all polyclonal
antibodies from Santa Cruz Biotechnology, CA), anti-parvalbumin (1:1000, Sigma) or
anti-COX-1 (1:100, Cayman Chemicals, cat. no. 160110). After washing, coverslips were
exposed for 1 h at room temperature to donkey anti-goat antibodies (or goat anti-mouse
for anti-parvalbumin and anti-COX-1 antibodies) conjugated to the fluorophore Alexa
Fluor 594 (1:200, Invitrogen), washed three times in PBS/gelatin, and visualized.
Double staining for IB4, on coverslips previously processed for PKCε, was assessed
by incubating the cells for 1 h at room temperature with IB4 bound to Alexa Fluor
594 (1:100, Invitrogen) followed by washing (three times). Coverslips were stored
at 4°C in sodium azide (0.05% in PBS) for additional analysis. Non-neuronal cells
in DRG cultures were identified as glial by morphology by their lack of response to
25 mM KCL and by staining with the glial-specific anti-S100 antibody (Sigma) (not
shown).

Quantification of PKCε translocation

Activation of PKCε results in translocation from an entirely cytoplasmic location
to the neuronal cell membrane. Translocation was quantified by determining fluorescence
intensity along a line positioned across the cell so as to avoid the nucleus (for
details see Cesare et al, 1999). Neurones in which intensity at the cell membrane
was 1.5× greater than the mean of cytoplasmic intensity were counted as positive.

Intracellular calcium imaging

Calcium imaging was performed as described previously [32-34]. In brief, isolated DRG neurons, plated onto glass coverslips were loaded with the
calcium-sensitive fluorescent indicator Fluo-4 AM (10 μM; Invitrogen). Coverslips
were imaged with an inverted confocal microscope (MicroRadiance; Bio-Rad, Hemel Hempstead,
UK) or with a camera-based system (Andor Technology, Belfast, UK) in HBSS (140 mM
NaCl, 1.8 mM CaCl2, 1 mM MgCl2, 4 mM KCl, 10 mM HEPES, 4 mM glucose, pH 7.4). High numerical aperture 10× or 20×
objectives were used. PAR agonists used are given in Table 2 below. The peptide agonist TFLLR was used to activate PAR1 apart from in experiments
on intact rat skin, where the more effective rat agonist SFLLRN was used. Proteases
were purchased as 1,000 NIH units and concentrations were calculated from conversion
factors supplied by the manufacturer giving units/mg protein. Neurons were distinguished
from non-neuronal cells by applying 25 mM KCl, which induces a rapid increase of [Ca2+]i only in neurons. At the end of the experiment, the maximal fluorescence (Fmax) was obtained by application of ionomycin (10 μM; Calbiochem, La Jolla, CA) in the
presence of Ca2+ (30 mM) and K+ (125 mM). Data are expressed as ΔF/Fmax. All experiments were performed at 20-22°C (RT).

The protocol used for sensitisation experiments was similar to that previously described
[43]. In brief, cells were exposed to short (1.6 s) repeated capsaicin applications, and
PAR activator peptides or proteases (Table 2) were applied for 2 min prior to the sixth capsaicin application. For control experiments
PAR activator application was omitted. The ratios obtained by dividing the amplitude
of the sixth capsaicin peak by the amplitude of the fifth capsaicin peak were used
to plot a histogram for each treatment group. The distribution seen for each group
was compared with the distribution obtained from control, vehicle-treated cells. A
neuron was defined as sensitized if the ratio was greater than the upper 99.7% confidence
interval calculated from control neurones. Ad-hoc software was written for analysis
of calcium imaging traces.

CGRP release studies

Male Wistar rats (80 - 100 g) were sacrificed in CO2 and the hairy skin of the hindpaw (26 ± 14 g, mean ± SD) was subcutaneously excised
from the knee to the foot sparing larger vessels and nerves. The skin flap was wrapped
around an acrylic rod with suture thread, exposing the corium side, and the preparation
was placed in a beaker containing physiological buffer solution bubbled with 95% O2, 5% CO2, pH 7.4 in a water bath (32°C) for 30 min to equilibrate. The skin sample was then
sequentially advanced in 5 min intervals through a series of six glass test tubes
filled with 1.2 ml gassed buffer and mounted in a shaking bath. The first tube was
to measure basal CGRP secretion, the subsequent three tubes (15 min) contained the
PAR1 or PAR2 activator peptide (table 2) or buffer solution for control, the fifth tube was at a temperature of 47°C to apply
noxious heat stimulation, and the sixth and final tube (again 32°C) was to measure
recovery or residual CGRP release. Each incubation fluid was immediately processed
to determine the CGRP concentration (pg/ml) using a commercial enzyme immunoassay
kit according to the manufacturer's instructions (SPIbio, Montigny, France). The procedure
has previously been validated and described in detail [44].

Statistical analysis

Statistical comparisons were performed with one-way analysis of variance (ANOVA),
followed by Bonferroni or Scheffé post hoc test and χ2 (SPSS for windows); pairwise comparisons were made using Student's t-test.

Results

Expression of PARs in DRG neurons: in situ hybridisation

In situ hybridization (ISH) was implemented to determine the cellular distribution
of PAR subtype mRNAs in DRGs from adult mice as previously described [40]. In Fig. 1A, bright-field photomicrographs of representative examples of autoradiographs show
the localization of oligonucleotide probes complementary to mouse PAR1, 2, 3, 4 mRNA
(arrowheads). Silver grains are visualised as small black dots overlying tissue sections.
Probes with comparable activity were used for each receptor. PAR3 mRNA was intensely
expressed in many DRG neurons and the mRNAs for PAR1, PAR2 and PAR4 were more weakly
expressed. A comparison of expression intensity between receptor subtypes is subject
to several variables, such as efficiency of the labelling reaction to incorporate
35S-dATP tails into the oligonucleotide probes, and hybridisation strength, though
these effects were minimised as much as possible. However, within these limitations
it seems clear that the expression of PAR3 is much more intense than that of PAR1,
2 and 4. In the case of PAR1, 2 and 4 a quantitative method was used to distinguish
neurons with signal above background, see Methods and [40].

Figure 1.Expression of PAR1-4 in sections of adult mouse DRG. A. In situ hybridisation (ISH) for PAR1-4 carried out as described in Methods. Positive cells
shown by arrowheads. Sections counterstained using hematoxylin-eosin. Scale bars 40
μm.

B. Similar sections in which PAR1, 3 and 4 expression was determined using immunohistochemistry.
Positive cells shown with arrows. The PAR2 antibodies available to us were found to
be non-specific on Western blots and results for PAR2 are therefore not shown. Sections
counterstained using hematoxylin-eosin. Scale bars 40 μm.

C. Expression of PAR1 - 4 as a function of neuronal size in adult mouse DRG using
ISH. Overall neuronal population (grey) is compared with those positive for each PAR
isoform (black). Overall, PAR1 was found to be expressed in 15.0% of neurones, PAR2
in 21.5%, PAR3 in 49.5% and PAR4 in 14.5%.

D. Similar results obtained using immunohistochemistry. PAR2 is not shown because
the antibody was found to exhibit non-specific binding, and PAR4 is not shown because
it proved impossible to distinguish neuronal from glial cell staining (see B). Overall
PAR1 was expressed in 10.3% of neurones, and PAR3 in 42.0%.

Fig. 1C shows histograms of cells positive for PAR1-4 against mean cross-sectional area of
neuronal profiles (between 912 and 1072 cells obtained from five sections for each
PAR and from three adult animals). Grey histograms indicate all neuronal profiles
with nuclei present that were measured, and black bars show profiles with a positive
in situ hybridization signal/noise ratio. PAR1 mRNA was found to be expressed in 15.0
± 1.5% of DRG neurones across all size classes. PAR2 mRNA was present in 21.5 ± 3.4%
of total neuronal profiles, almost exclusively in neurones with a small cross-sectional
area; there was only a low level of mRNA expression in medium sized neurones and no
detectable expression in neurones with a large cross-sectional area. PAR3 was present
in 49.5 ± 4.5% of neurons and was expressed mainly in neurones with a small cross-sectional
area, but unlike PAR2, PAR3 mRNA was also expressed in medium-sized neurones. PAR4
mRNA expression was found in a similar proportion of neurones to PAR1 (14.5% ± 4.3)
and with a distribution of expression across the neuronal size range similar to that
found for PAR1.

Expression of PARs in glia was difficult to detect unequivocally using ISH because
of the small size of these cells and the scatter of silver grains. We show below that
there is clear functional expression of PAR1 and PAR2 in glial cells.

Expression of PARs in DRG neurons: immunohistochemistry

PAR1, 3 and 4 immunoreactivity (IR) was detected in DRG neurons (Fig. 1B). The PAR2 antibodies available to us showed clear evidence of non-specific bands
on Western blot and results are therefore not shown. Preabsorption controls with a
20-fold excess of immunising peptide completely ablated the signals for PAR1, 3 and
4 in adjacent sections, as did incubation in the absence of primary antibody (data
not shown). As with ISH, the distribution of expression was determined by measuring
neuronal cross-sectional area, and by only including profiles in which there was a
visible nucleus (Fig. 1D).

PAR1-IR was restricted to a small percentage of neurones (10.28 ± 2.54%) and was expressed
in cells across the neuronal size range. The results for PAR1-IR were similar to those
obtained with ISH (compare Fig. 1C and 1D). Expression was punctate and appeared particularly intense in vesicular structures
surrounding the nucleus, suggesting the presence of large intracellular stores of
protein. PAR3-IR was detected in 42.03 ± 4.95% of neuronal profiles (Fig. 1D), similar to results obtained using ISH (Fig. 1C). PAR4-IR was also detected in DRG neurons but PAR4-IR was particularly strongly
expressed in glial cells, and it was not always possible to distinguish positive neurones
stained at the plasma membrane from surrounding ensheathing glial cells (see Fig.
1B). For this reason PAR4-IR was not quantified.

Calcium signals activated by PAR agonists in DRG neurons

We next examined functional activation of PAR receptors. A sub-population of small
DRG neurons responded to the specific PAR2 activator peptide SLIGRL (PAR2-AP), which
is derived from the activator domain of PAR2 (Fig. 2A). The proportion of neurons responding to the PAR2-AP with an increase in [Ca]i was 15.6% in neonatal rats and 12.1% in neurons from adult mice. A large majority
of the PAR2-AP responsive neuronal population also expressed TRPV1 and TRPA1, as shown
from the increase in [Ca]i in response to the specific TRPV1 agonist capsaicin and to the specific TRPA1 agonist
mustard oil, and bound the plant isolectin B4 (IB4), which identifies a non-peptidergic
subpopulation of nociceptors (see Fig. 2A and 2B). These PAR2+ neurons therefore have the characteristics of IB4-positive nociceptors. None of these
neurons, however, responded to thrombin and so are unlikely to express PAR1 or 4 (PAR3
appears unresponsive in DRG neurons, see below).

Figure 2.Calcium signals elicited by PAR agonists. A. Adult mouse neuron in which an increase in [Ca]i was elicited by a specific PAR2 activator peptide (PAR2-AP, SLIGRL, 100 μM), but not
by thrombin (100 nM) which activates PAR1, 3 and 4. The neuron also expresses receptors
for TRPA1 and TRPV1, as shown by its responses to the specific TRPA1 agonist mustard
oil (MO, 100 μM) and the specific TRPV1 agonist capsaicin (1 μM).

C. Adult mouse neuron in which an increase in [Ca]i was elicited by thrombin (100 nM). This neuron also expresses the ion channels TRPA1
and TRPV1, as shown by its responses to mustard oil (MO, 100 μM) and capsaicin (1
μM). Cell was identified as a neuron on morphological grounds, confirmed by calcium
increase observed in response to 25 mM KCl.

E. Glial cell which responded with increase in [Ca]i to PAR1-AP (100 μM) and to PAR2-AP (100 μM). Cell was identified as a glial cell on
morphological grounds, confirmed by absence of calcium increase in response to 25
mM KCl. In separate experiments, cells of this morphology were also identified by
the glial-specific anti-S100 antibody (not shown).

F. Percentage of glial cells responding to thrombin (100 nM), PAR1-AP (100 μM) and
PAR2-AP (100 μM). Deletion of PAR1 ablated responses to both thrombin and PAR1-AP
(bars 4 and 5) while deletion of PAR2 was without effect on responses to thrombin
and PAR1-AP (bars 6 and 7).

Thrombin, which activates PAR1, 3 and 4, elicited robust increases in [Ca]i in a distinct sub-population of sensory neurons (Fig. 2C,D). The proportion of neurons responding to thrombin with an increase in [Ca]i was 17.5% in neonatal rats and 15.2% in neurons from adult mice. No neuron responsive
to thrombin also responded to PAR2-AP (Fig. 2B,D). Among these thrombin-responsive neurons, around 25-33% responded to capsaicin,
mustard oil, and to the peptides Bv8 and bradykinin, both of which act on G-protein
coupled receptors expressed in nociceptors [34], but none bound IB4 (Fig. 2D). About a third of thrombin-responsive neurons are therefore IB4-negative nociceptors,
while the remainder are non-nociceptive. As PAR2 is predominantly expressed in IB4+ nociceptors (see above) this shows that functional PAR2 and PAR1/3/4 receptors are
located in separate subpopulations of nociceptors. PAR4 was found to be colocalised
with PAR1 expression in neonatal rat neurons, because calcium responses to a PAR4-AP
(AYPGKFR) were elicited in a subset of PAR1 expressing neurons (see below) and all
cells responding to PAR4-AP also exhibited a calcium signal in response to PAR1-AP
(not shown).

Glial cells are clearly distinguishable from neurons both on morphological grounds
and because they do not exhibit a calcium increase in response to elevated [K+] (Fig. 2C). Most glial cells responded to thrombin (Fig. 2E). A few glial cells responding to thrombin also responded to PAR2-AP (3.6% -see Fig.
2E,F), showing that in contrast to neurons, PAR2 and PAR1/3/4 are co-expressed in a small
subset of glial cells. The calcium response to thrombin and PAR1-AP in glial cells
was ablated by genetic deletion of PAR1 but was unaffected by deletion of PAR2 (Fig.
2E).

PAR3 is highly expressed in DRG neurons (Fig. 1). PAR3 mRNA is seen in about 50% of neurons and PAR3 protein is seen in about 42%
of neurons. Functional responses to thrombin, which should activate PAR3 (along with
PAR1 and PAR4), are seen in a significantly lower number of neurons, however, suggesting
that PAR3 is non-functional, at least when expressed in the absence of other PARs.
To test this more conclusively it would be desirable to activate PAR3 alone, but specific
activation of PAR3 in neurons coexpressing PAR1 and PAR4 is not possible because PAR3
peptides also activate PAR1 and PAR4 [45,46]. We therefore tested for PAR3 responses by desensitizing PAR1 and 4 with their specific
activator peptides, and then retesting with thrombin (Fig. 3). Following desensitization of PAR1 and PAR4, calcium signals in response to thrombin
are seen in only a very small number of neurons, far smaller than the proportion in
which histological studies had shown expression of PAR3. These results support the
idea the PAR3 is largely non functional by itself in DRG neurons, but they do not
rule out the possibility that PAR3 may heteromerise with other PARs to form functional
receptors, as has been found in other studies [47,48].

Figure 3.Desensitization of PAR1 and PAR4 ablates calcium signals in response to thrombin. A. Increase in [Ca]i recorded as in Fig. 2. Calcium increase elicited by application of PAR1-AP completely
desensitizes response to a subsequent application of PAR1-AP but not to PAR4-AP. The
calcium signal in response to thrombin was ablated in the large majority of cells
by desensitization of both PAR1 and PAR4. All experimental details as in Fig. 2.

B. Following desensitization of PAR1 and PAR4 only 1.6% of neurons gave a calcium
signal in response to thrombin, compared with 15.2% in control neurons. Summary of
results from n = 187 neurons from 4 separate coverslips.

Sensitization of TRPV1 by PAR activation

Activation of the heat and capsaicin gated ion channel TRPV1 is potentiated by PAR2
activation [22]. Fig. 4 shows a similar potentiation of heat-activated inward currents by specific PAR1 and
PAR4 activator peptides. Both PAR agonists caused substantial sensitization of TRPV1
in a subset of neurons (c. 10% of total neurons, consistent with studies of expression
of PAR1 or PAR4, see above). Sensitization was long-lasting and subsequent PAR-AP
applications were ineffective (Fig. 4B,D). Thrombin and trypsin also caused a substantial enhancement in the inward current
activated by heat (Fig. 4E).

Figure 4.Sensitization of TRPV1 by PAR activation. A - D. Heat-activated currents were significantly enhanced in c. 10% of neurons
by application of PAR1-AP and PAR4-AP. Single traces in panels to left are taken from
time courses shown in right hand panels. Both PAR1-AP (TFLLR at 100 μM) and PAR4-AP
(AYPGKF, 200 μM) caused long-lasting sensitisation. Sensitization showed complete
tachyphylaxis on a second application.

E. Percentage sensitization in experiments similar to those in A. Thrombin (100 nM),
trypsin (100 nM), PAR1-AP and PAR4-AP all caused approximately a doubling of the inward
current elicited by heat. Thrombin-induced sensitisation was largely blocked by the
PKC inhibitor Ro318220 (1 μM) and by the broad-spectrum kinase inhibitor staurosporine
(1 μM, both applied throughout the experiment).

Many pro-inflammatory mediators sensitize TRPV1 via downstream activation of PKCε,
reviewed in [49]. Consistent with this also being the principal signaling pathway activated by PAR1/3/4,
Fig. 4E shows that the sensitization caused by thrombin was reduced at least 5-fold by the
specific PKC inhibitor Ro-318220 or by the broad-spectrum kinase inhibitor staurosporine.

We next tested sensitization of TRPV1 by thrombin in wild-type and PAR1-/- mice. In order to improve cell yield we employed a calcium imaging protocol similar
to that used by Bonnington & McNaughton [43]. We activated TRPV1 by applying brief pulses of the specific agonist capsaicin, and
tested the effect of thrombin in enhancing TRPV1 activation (Fig. 4F). Ratios of responses to capsaicin before and after application of thrombin were
calculated, and sensitized cells were identified when the ratio exceeded the 99.7%
confidence limits of a distribution obtained from control experiments (see Additional
file 1). In PAR1-/- animals the percentage of sensitized neurons using thrombin as a PAR activator was
8.3%, significantly lower than in WT neurons (Fig. 4G). Thus removal of PAR1 reduces but does not completely abolish the response to thrombin,
consistent with the idea (see above) that DRG neurons also express functional PAR4
receptors.

Note that the percentage of neurons from PAR1-/- mice responding to thrombin is similar to the proportion of neurons expressing PAR4
by ISH (14.5%, see Fig. 1C above) but is very much smaller than the proportion expressing PAR3 by both ISH and
immunohistochemistry (49.5% and 42.03% respectively, see Fig. 1C,D above). Combined with the results in Fig. 3 (above) these results suggest that PAR4 receptors in DRG neurons are functionally
activated by thrombin but that PAR3 receptors are not.

PAR1/4 agonists cause translocation of PKC-ε in sensory neurons

The activation of PKCε can be visualized as a translocation from the cytoplasm to
the cell surface membrane, and provides a sensitive indicator of those neurons activated
by bradykinin [21,33] or by other pro-inflammatory mediators [34]. We found that thrombin and PAR1-AP caused a pronounced translocation of PKC-ε to
the neuronal cell membrane in a subset of neurons from adult and neonatal rats and
from adult mice (Fig. 5A). PKCε translocation, expressed as the percentage of neurons in which clear translocation
was observed, peaked at 30 s after application of a maximal concentration of 100 nM
thrombin (Fig. 5B). At longer application times PKCε was internalized into peri-nuclear vesicles (Fig.
5A, right hand panel), as is seen after longer exposures to bradykinin [21] and to the prokineticin receptor agonist Bv8 [34]. Translocation of PKCε was half-activated by a concentration of 2.0 ± 0.4 nM thrombin
and was fully saturated at 100 nM thrombin (Fig. 5C). In adult mouse neurons cultured without NGF translocation was observed in 15.6
± 0.5% of the population (Fig. 5D), a proportion which increased to 19.3 ± 1.0% with NGF (see below). Responsive neurons
were distributed across all neuronal size classes, in agreement with histological
data for expression of PAR1 and 4 (Fig. 1).

Figure 5.Translocation of PKCε to neuronal surface membrane caused by thrombin. A. Translocation of PKCε to neuronal surface membrane in control conditions (left)
and following exposure to thrombin (100 nM, 30 and 60 sec). PKCε translocated rapidly
to the surface membrane following application of thrombin (arrow in middle panel)
and at longer times became progressively internalised (arrowhead in middle panel and
right panel). Adult mouse neurons cultured in 10% FBS in absence of NGF and neurturin.
Scale bars 5 μm.

B. Percentage of neurons showing translocation to the plasma membrane as a function
of time of exposure to thrombin (number of neurons > 2000 for each point).

C. Peak percentage of neurons in which PKCε was translocated, as a function of thrombin
concentration (number of neurons > 2000 for each point). Continuous curve shows a
Hill equation with n = 0.7 and K1/2 = 2 nM.

Other proteases known to activate PAR1 and PAR4 were also effective in causing translocation
of PKCε (Fig. 5E). Trypsin, a broad-spectrum PAR activator, produced translocation in 17.1 ± 1.6%
of neurons. Cathepsin G, which preferentially activates PAR4 over PAR1, caused translocation
in 11.8 ± 2.3% of neurons. Type IV collagenase was ineffective. The PAR1-AP TFLLR
caused translocation in a similar proportion of neurons to thrombin, while the specific
PAR4-AP AYPGKF caused translocation in a significantly lower percentage of neurons
than thrombin (9.1 ± 2.1%, n = 5 p < 0.05). Application of the PAR1-AP TFLLR in combination
with PAR4-AP gave only a slightly higher percentage than PAR1-AP applied alone (17.3
± 0.6%, n = 6). These data agree with those above (Fig. 3B) in showing that PAR4 receptors are expressed in a subset of the PAR1-expressing
sensory neurons. PAR2-AP was ineffective in causing translocation of PKCε in any neuron.

Characteristics of neurons expressing functional thrombin receptors

We next examined the histological characteristics of thrombin-responsive neurons,
using translocation of PKCε as a marker. Around half of the thrombin-responsive neuronal
population, predominantly medium-sized and large neurons, stained for neurofilament
H (NFH+), a marker for myelinated neurons (second bar in Fig. 6B). Only a small fraction (around 6%) of these NFH+ neurons also expressed functional TRPV1 receptors, as demonstrated by a calcium increase
in response to application of capsaicin (see white bar at bottom of second bar in
Fig. 6B), showing that this class of thrombin-responsive large neurons is predominantly non-nociceptive.

Figure 6.Co-localisation of thrombin-induced translocation of PKCε with other neuronal markers. A. PKCε translocation (green) following exposure to thrombin (100 nM, 30 s) colocalises
with other neuronal markers as shown. PKCε translocation was co-localised in c. half
of cells with expression of the neuropeptide CGRP and with the neurofilament marker
N52, and in a smaller proportion of cells with the neuropeptide substance P (SP) (panels
on right). PKCε translocation was not in general co-localised with IB4 binding nor
with parvalbumin (Prv) or COX-1 (panels on left). Neurones from adult mice cultured
in the absence of NGF, with the exception of the COX-1 experiment which was carried
out in neonatal rat sensory neurons cultured in NGF (100 ng/ml) as the antibody available
to us did not bind mouse COX-1. Scale bars all 5 μm.

B. Summary of results from experiments similar to those shown in A. First bar shows
percentage of cells showing translocation of PKCε in response to thrombin (100 nM,
30 s). Remaining bars show percentages of these thrombin-responsive cells which co-expressed
the neuronal markers noted beneath each bar. White bar in N52 column shows the proportion
of the N52 positive neurons in which TRPV1 expression had been demonstrated by recording
a calcium increase in response to capsaicin prior to fixation (c.f. Fig. 2). Final
white bar shows overall fraction of thrombin-responsive neurons in which TRPV1 expression
had been demonstrated by calcium imaging.

A distinct population of thrombin-responsive neurons, mainly small neurons, co-expressed
the neuropeptides CGRP and/or substance P (bars 3 and 4 in Fig. 6B). Neurons in this population gave a calcium increase in response to capsaicin and
therefore express TRPV1 (final bar in Fig. 6B). Very few thrombin-responsive small neurons were IB4-positive (5% of the thrombin-responsive
population), in agreement with Fig. 2D above where neurons responding to thrombin with a calcium increase were found to
be IB4-negative. The presence of neuropeptides and the lack of binding of IB4 identifies
a TrkA positive C-fibre nociceptor sub-population [50] as the location of nociceptor PAR1/4 expression. In addition, thrombin-responsive
neurons were negative for cyclooxygenase 1 (COX-1), an enzyme expressed in a subpopulation
of small-sized nociceptive neurons [51], and for parvalbumin, expressed in non-nociceptive sensory neurons innervating muscle
spindles [52]. In summary, our data show that functional receptors for thrombin are expressed broadly
across all neuronal size classes, in neurons subtending both myelinated and unmyelinated
fibres. In the unmyelinated neuronal population thrombin-responsive neurons are found
in the peptidergic/IB4- class of nociceptors.

Release of CGRP by heat is potentiated by PAR1

The results outlined above show that PAR1/4 receptors in small neurons co-express
with TRPV1 and the neuropeptide CGRP, suggesting that neuropeptide release caused
by TRPV1 activation should be potentiated by PAR1. Fig. 7 shows an experiment in which this hypothesis was tested using a rat skin preparation,
which contains nerve terminals from which CGRP can be released by noxious heat stimulation
[53]. In mouse skin, this heat response is markedly reduced, though not abolished, if
the TRPV1 gene is deleted, and it is sensitized by pre-treatment with the weak TRPV1/2/3
agonist 2-APB which is ineffective in TRPV1 knockouts [54]. In the present experiments, the heat stimulation caused about a tenfold increase
in CGRP release from cutaneous nerves (p < 0.001, t-test). The basal CGRP release
was unaffected by the presence of PAR1-AP. The release in response to heat was approximately
doubled by exposure to the PAR1-AP, consistent with expression of PAR1 in peptidergic
neurons.

Upregulation of PAR expression by neurotrophins

Fig. 8 examines the effect of exposure to neurotrophic factors on expression of functional
PAR1/4 receptors, measured from PKCε translocation following exposure to thrombin.
NGF and neurturin (NTN) applied individually significantly increased the number of
thrombin-responsive small neurons, while the effects of NGF and NTN applied together
were additive, consistent with the known expression of TrkA and Ret receptors in separate
neuronal populations (Fig. 8A). In the absence of neurotrophins few thrombin-responsive neurons bind IB4 (first
bar in Fig. 8A). NGF increased the proportion of thrombin-responsive neurons but IB4 binding was
not significantly increased. NTN, on the other hand, significantly upregulated the
proportion of the thrombin-responsive population stained by IB4.

B. Deletion of PAR1 reduces but does not eliminate responsiveness to thrombin.

C. Deletion of PAR2 does not affect proportion of neurons responsive to thrombin.

In neurons from PAR1-/- animals the proportion showing PKCε translocation was significantly reduced but was
not zero (Fig. 8B), consistent with an action of thrombin on PAR4 as discussed above. The fraction
of responsive neurons was upregulated by NGF and NTN. In neurons from PAR2-/- animals the proportion of neurons activated by thrombin, and the effects of NGF and
NTN in upregulating the proportion of thrombin-responsive neurons, were similar to
wild-type neurons (Fig. 8C), confirming that PAR2 receptors are not involved in responses to thrombin.

Discussion

The work described here demonstrates a previously unsuspected role for PAR1 and PAR4
protease-activated receptors in nociceptive neurones. We used both histological and
functional expression studies to explore the expression of PAR receptors in sensory
neurons. For many of the functional studies we used thrombin, a protease which activates
PAR1, 3 and 4 but not PAR2 receptors. In initial studies we used neurons from mice
and rats, and from both adult and neonatal animals, in order to gain an understanding
of how responses to PAR agonists differ across species and at different ages. In fact
there were few significant differences (see Table 3) and we therefore focussed on adult mice in the majority of experiments in order
to compare our results with those from PAR knockout animals.

Table 3. Summary of characteristics of DRG neurons from adult and neonatal mice and rats

We find clear evidence for expression of PAR1/4 receptors in a population of peptide-expressing,
IB4-negative nociceptive neurones, where they couple to PKCε, cause sensitization
of TRPV1 and promote the heat-dependent release of the pro-inflammatory neuropeptide
CGRP. These observations suggest a role for PAR1/4 receptors in promoting inflammation
and pain following the release of thrombin. Functional PAR1/4 receptors are also found
in a fration of large diameter neurones which express neurofilament H and would therefore
in vivo subtend myelinated fibres. Only a small minority of these NFH+ neurons express TRPV1 (Fig. 6B), suggesting that most serve a non-nociceptive function.

PAR1

PAR 1 is expressed in around 15% of primary sensory neurons from adult mice. Consistent
data were obtained from in situ hybridisation (15%, Fig. 1C), from immunohistochemistry (10%, Fig. 1D), in functional studies from sensitization of the capsaicin response (15%, Fig. 3G) and from translocation of PKCε (15.6%, Fig. 5B). These measures also showed that PAR1/4 expression was distributed approximately
equally across all neuronal size classes (Fig. 1 and Fig. 5D). In agreement with this, approximately half of the neurons responding to thrombin
were positive for neurofilament H, a marker for larger neurons subtending myelinated
fibres (Fig. 6B). Most of the remainder of the thrombin-responsive neurons (c. one third of the total)
were small and expressed functional TRPV1, TRPA1 and prokineticin and bradykinin B2
receptors (Fig. 2) and contained neuropeptides (Fig. 6B), all of which are characteristic of the small and medium-sized nociceptive neuronal
population. The myelinated-fibre and nociceptor PAR1-expressing populations are mostly
distinct, because only a small fraction of neurons expressing neurofilament H also
express TRPV1 (Fig. 6B). Thus the PAR1-expressing neuronal population can be divided in broad terms into
two functionally distinct classes: myelinated-fibre neurons, most of which do not
express TRPV1; and unmyelinated-fibre neurons expressing neuropeptides, TRPV1 and
other markers for nociceptors. Dai et al [16] found that PAR1 was not co-expressed with TRPV1 and therefore would not be expected
to play a role in nociception, but in the present study we find by using several independent
approaches that there is strong evidence for co-expression of functional PAR1 receptors
and TRPV1 in the peptidergic subset of small and medium-sized neurones.

Several observations show that functional PAR1 and PAR4 receptors are not expressed
in the non-peptidergic, IB4-positive class of nociceptors. Few thrombin-responsive
neurons were IB4-positive (Fig. 2 and Fig. 6B), and small thrombin-responsive neurons express the neuropeptides CGRP and SP (Fig.
6) which are known not to be colocalized with IB4 binding. Finally, exposure to NGF
increases the fraction of thrombin-responsive neurons (Fig. 8A), implying the presence of functional TrkA receptors, which are known to be expressed
in the IB4- population.

One important functional consequence of PAR1/4 activation in nociceptive neurons is
that both receptors can sensitize the heat and capsaicin receptor, TRPV1, which in
vivo has been shown to produce a state of heat hyperalgesia [55]. The membrane current carried by TRPV1 in response to either heat or capsaicin was
approximately doubled in responsive neurons following exposure to thrombin or PAR1
and PAR4 activator peptides (Fig. 4). PAR receptors couple to Gq, leading to activation of protein kinase C [5] which has in turn been shown to phosphorylate and sensitize TRPV1, as reviewed in
[49]. Most of the sensitization of TRPV1 by PAR1 is abolished by PKC inhibitors (Fig.
3B), showing that phosphorylation by PKC is also the main pathway important in sensitization
of TRPV1 by PAR1/4.

The sensitization of TRPV1 by thrombin, together with the observation that CGRP is
expressed in the thrombin-responsive nociceptor population, suggests that the CGRP
release activated by heat should be enhanced by PAR1 activation. This prediction was
borne out in experiments performed on isolated rat skin containing intact peptidergic
nerve terminals; the heat-dependent CGRP release was strongly potentiated by PAR1
activation (Fig. 6). The implication of this experiment is that PAR1 activation should play a role in
potentiating neurogenic inflammation, in which neuropeptides such as CGRP are released
from nociceptive nerve terminals following noxious insults or cell damage.

PAR2

The role of PAR2 in sensory neurones has been explored fully in studies from other
labs and was examined in less depth in the present study than that of PAR1/4. In situ
hybridization (Fig. 1) showed that PAR2 is expressed almost exclusively in the small neuronal population,
the majority of which are nociceptors, as was found by Amadesi et al [17]. In agreement with this, c. 80% of PAR2-AP responsive neurones expressed functional
TRPV1 and TRPA1 ion channels (Fig. 2B). One surprise, though, in view of previous studies implicating PAR2 in neuropeptide
release [13,15] was that the large majority of neurones in which PAR2-AP elicited a calcium signal
were IB4-positive, and therefore belong to a population which is predominantly non-peptidergic
(Fig. 2B).

PAR3

PAR3 was strongly expressed, mainly in small neurones (Fig. 1). The percentages of neurones expressing PAR3 determined by in situ hybridization
and immunohistochemistry were in good agreement (49% and 42%, respectively). However,
when thrombin or trypsin, both of which activate PAR3 along with PAR1/4, were used
in a number of different studies of functional expression, responses were seen in
a significantly smaller proportion of neurones than those suggested by histological
data for PAR3. Desensitization of PAR1/4, which should leave PAR3 unaffected, in fact
largely ablated the response to thrombin (Fig. 3). Thus PAR3 must either be non-functional in sensory neurones, or else is only able
to act in concert with other PAR receptors, as has been noted in other studies [see
47;48].

PAR4

PAR4 expression, like PAR1, was found by in situ hybridization to be broadly distributed
across all neuronal sizes (Fig. 1). There is clear evidence for a functional role for PAR4 in sensory neurons. PAR4-AP
was found in patch clamp experiments to cause a sensitization of TRPV1 as potent as
that of PAR1-AP, although in fewer neurons, and in calcium imaging experiments the
percentage of cells sensitized by thrombin was reduced by less than half, from 15%
to 8%, by genetic deletion of PAR1 (Fig. 4). Consistent with this, PAR1-AP caused translocation of PKCε in c. 15% of neurons,
while PAR4-AP caused translocation in 9% (Fig. 5). PAR4 is expressed only in PAR1-expressing neurons, because all neurons responding
to PAR4-AP also responded to PAR1-AP (Fig. 3). PAR2, by contrast, is expressed in a distinct neuronal subpopulation (Fig. 2B,D).

Upregulation of PAR expression by neurotrophins

Neurotrophins enhance the sensation of pain partly by upregulating a wide variety
of proteins important in nociception. We have shown that both NGF and neurturin upregulate
thrombin-responsiveness in sensory neurones (Fig. 7). The increase in responsiveness was seen as an increase in the number of neurons
expressing functional PAR1/4 receptors in response to a maximal dose of thrombin,
suggesting the de novo appearance of functional receptors in neurons that previously
did not express them, rather than sensitization of existing receptors. The action
is on the small neurone population (Additional file 2), in agreement with the known expression of both TrkA and Ret in small nociceptive
neurons. The results are consistent with an action of the two neurotrophins on separate
neuronal populations, however, because NGF does not increase the few IB4+ neurones which respond to thrombin, while NTN does increase the number of these IB4+ neurons, consistent with the idea that NGF unregulates the number of PAR1/4 expressing
neurons in the peptidergic population, while NTN induces de novo expression of PAR1/4
receptors in the IB4+ neuronal population.

Functional implications

Activation of PAR2 receptors is well known to cause inflammation [19] but a role for PAR1 and 4 is less clear. Previous studies have shown that PAR1/4
activation has a dual role: low doses are antinociceptive, while higher levels cause
inflammation and pain [3,28,56,57]. The finding in the present study that PAR1/4 receptors are expressed in two distinct
populations of sensory neurons suggests a possible basis for this dual effect. Activation
of large-diameter myelinated afferents is well known to have an antinociceptive effect,
and the activation of PAR1/4 in these afferents could therefore have an analgesic
action. The expression of PAR1/4 in small-diameter nociceptive afferents, on the other
hand, where they can potentiate TRPV1 and enhance the release of neuropeptides, provides
a ready explanation for the inflammatory effects of higher levels of thrombin and
specific PAR1/4 agonists. Following injury and rupture of blood vessels the release
of significant amounts of thrombin could act on nociceptive nerve terminals, sensitizing
TRPV1 to heat stimuli and promoting the release of pro-inflammatory neuropeptides
such as CGRP, as has been shown in this study. Thus we propose that higher levels
of thrombin can act in a similar way to other better-studied pro-inflammatory mediators,
in promoting neurogenic inflammation and heat hyperalgesia in injured tissues through
the sensitization of TRPV1.

Conclusions

In summary, we find clear evidence for co-expression of functional PAR1 and PAR4 receptors
in a sub-population of small peptide-expressing nociceptive neurones, where they couple
to PKCε, cause sensitization of TRPV1 and promote the heat-dependent release of the
pro-inflammatory neuropeptide CGRP. This study therefore suggests a previously unsuspected
role for PAR1 and PAR4 in mediating the inflammation and pain caused by tissue damage
severe enough to rupture blood vessels. Functional PAR1/4 receptors are also expressed
in large diameter myelinated-fibre neurones which do not express TRPV1 and are therefore
likely to be non-nociceptive. The role of PAR1/4 in these non-nociceptive neurones
is less clear, but they may be responsible for the antinociceptive effects of low
concentrations of thrombin.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

VV designed and carried out all experiments except those in Fig. 1 and Fig. 7, and wrote the first draft of the manuscript. AMK designed and carried out the histological
experiments in Fig. 1, contributed to the Ca imaging experiments and commented on the manuscript. MP carried
out calcium imaging and immunocytochemistry experiments on DRG cultures from transgenic
animals, carried out data analysis and statistical analysis, participated in experimental
design and in manuscript preparation. SH carried out the experiments in Fig. 7. PR designed the experiments in Fig. 7 and commented on the manuscript. PCM participated in experimental design and in manuscript
preparation. CG participated in experimental work, equipment design and data analysis
of electrophysiology experiments, carried out statistical analysis, wrote equipment
software and image and data analysis software necessary for the work, and participated
in manuscript preparation. PAM designed the project, advised on experiments, analyzed
and interpreted data and wrote the final version of the manuscript, which was approved
by all authors.

Acknowledgements

This work was funded by a project grant from the Wellcome Trust (to PAM), by an MRC
studentship (to AK), and by grants from Fondazione Cassa di Risparmio di Modena, Fondazione
Cassa di Risparmio di Carpi and an Italian MIUR grant 2004057339_002 (to VV).